Recombinant vesiculoviruses and their uses

Abstract

The present invention provides recombinant replicable vesiculoviruses. The invention provides a method which, for the first time, successfully allows the production and recovery of replicable vesiculoviruses, as well as recombinant replicable vesiculoviruses, from cloned DNA, by a method comprising expression of the full-length positive-strand vesiculovirus antigenomic RNA in host cells. The recombinant vesiculoviruses do not cause serious pathology in humans, can be obtained in high titers, and have use as vaccines. The recombinant vesiculoviruses can also be inactivated for use as killed vaccines.

1 - 43 . (canceled)  
     
     
         44 . A kit comprising in one or more containers (a) a first recombinant nucleic acid that can be transcribed in a host cell to produce an RNA molecule comprising a vesiculovirus antigenomic (+) RNA, in which a foreign RNA sequence is inserted into or replaces a region of the RNA nonessential for replication of the vesiculovirus (b) a second recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus N protein; (c) a third recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus L protein; and (d) a fourth recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus P protein.  
     
     
         45 . A kit comprising in one or more containers (a) a first recombinant nucleic acid that can be transcribed in a host cell to produce an RNA molecule comprising a vesiculovirus antigenomic (+) RNA; (b) a second recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus N protein; (c) a third recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus L protein; and (d) a fourth recombinant nucleic acid encoding and capable of expressing in the host cell a vesiculovirus P protein.  
     
     
         46 . A kit comprising in one or more containers (a) a recombinant nucleic acid that can be transcribed to produce an RNA molecule comprising a vesiculovirus antigenomic (+) RNA; and (b) a host cell that recombinantly expresses vesiculovirus N, P, and L proteins.  
     
     
         47 . The kit of  claim 45  in which said first recombinant nucleic acid further comprises a polylinker in a region that is transcribed to produce said RNA molecule, said region being nonessential for replication of the vesiculovirus.  
     
     
         48 . The kit according to  claim 45  in which the second recombinant nucleic acid is a DNA plasmid vector comprising the following operatively linked components: 
 (a) a promoter which controls the expression of the N protein;    (b) a translation initiation signal;    (c) a DNA sequence encoding the N protein; and    (d) a transcription termination signal; and    in which the third recombinant nucleic acid is a DNA plasmid vector comprising the following operatively linked components:    (a) a promoter which controls the expression of the P protein;    (b) a translation initiation signal;    (c) a DNA sequence encoding the P protein; and    (d) a transcription termination signal    and in which the fourth recombinant nucleic acid is a DNA plasmid vector comprising the following operatively linked components:    (a) a promoter which controls the expression of the L protein;    (b) a translation initiation signal;    (c) a DNA sequence encoding the L protein; and    (d) a transcription termination signal; and    in which the first recombinant nucleic acid is a DNA plasmid vector comprising the following operatively linked components:    (a) a promoter;    (b) a DNA sequence that can be transcribed under the control of the promoter in the cell to produce said RNA molecule, said RNA molecule further comprising a ribozyme sequence immediately downstream of said antigenomic (+) RNA, that cleaves at the 3′ terminus of the antigenomic (+) RNA, said DNA comprising a polylinker in a region that is transcribed to produce said RNA molecule, said region being nonessential for replication of the vesiculovirus; and    (c) a transcription termination signal.    
     
     
         49 . The kit of  claim 48  in which the promoter is an RNA polymerase promoter, and in which the cell also contains a cytoplasmic source of said RNA polymerase.  
     
     
         50 . A kit comprising 
 (a) in a first container a first DNA plasmid vector comprising the following operatively linked components: 
 (i) a bacteriophage RNA polymerase promoter;  
 (ii) a first DNA sequence that can be transcribed in a cell to produce an RNA molecule comprising (A) a vesiculovirus antigenomic (+) RNA in which a foreign RNA sequence is inserted into or replaces a region of the RNA nonessential for replication of the vesiculovirus, and (B) a ribozyme sequence immediately downstream of said antigenomic (+) RNA, that cleaves at the 3′ terminus of the antigenomic RNA; and  
 (iii) a transcription termination signal for the RNA polymerase;  
   (b) in a second container a second DNA plasmid vector comprising the following operatively linked components: 
 (i) the bacteriophage RNA polymerase promoter;  
 (ii) a second DNA sequence encoding an N protein of the vesiculovirus; and  
 (iii) a second transcription termination signal for the RNA polymerase;  
   (c) in a third container a third DNA plasmid vector comprising the following operatively linked components: 
 (i) the bacteriophage RNA polymerase promoter;  
 (ii) a third DNA sequence encoding a P protein of the vesiculovirus; and  
 (iii) a third transcription termination signal for the RNA polymerase;  
   (d) in a fourth container a fourth DNA plasmid vector comprising the following operatively linked components: 
 (i) the bacteriophage RNA polymerase promoter;  
 (ii) a fourth DNA sequence encoding an L protein of the vesiculovirus; and  
 (iii) a fourth transcription termination signal for the RNA polymerase; and  
   (e) in a fifth container a recombinant vaccinia virus comprising a sequence encoding the bacteriophage RNA polymerase.    
     
     
         51 - 74 . (canceled)